Clostridium difficile is a Gram positive, spore forming anaerobic bacillus, and many strains of this species have acquired resistance to a majority of commonly used antibiotics. The reduction of microflora as an effect of use of antibiotics allows C. difficile to grow and to produce harmful toxins in the intestine, without nutritional competition from normal bacterial flora. Highly virulent strains of C. difficile result in increased incidence of illness and more severe effects in patients.
C. difficile produces two major protein toxins, toxin A (TcdA) and toxin B (TcdB), which are 308 kD and 269 kD respectively in size. The two toxins belong to the large clostridial cytotoxin (LCT) family and share 49% amino acid identity. The toxins have similar structures and share putative receptor binding, transmembrane, and enzymatic domains. After receptor-mediated internalization and intracellular cleavage, the toxins glucosylate members of the Rho-Rac family of small GTPases at a specific threonine residue in host intestinal epithelial cells, leading to alterations in the actin cytoskeleton, massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa.
TcdB has been determined to be the critical virulence factor that contributes to illness, and is a target for diagnosis and vaccination. Neutralizing antibodies against toxin A and toxin B are thought to play an important role in the resolution of C. difficile-associated diarrhea (CDAD). Variations in these neutralizing responses, the absence of a neutralizing response, and the lack of cross-neutralization are all hypothesized to contribute to the range of disease severity and the potential relapse. By determining the specific shared and unique neutralizing epitopes, improved diagnostics can be designed (for disease severity, strain type, and relapse potential) as vaccines, and therapeutics for CDAD.
Several studies support the notion that antibodies to toxin A and toxin B protect against CDI. Most patients with acute infection do not exhibit an IgM response, but rather a secondary IgG response. In patients with acute C. difficile infection (CDI), higher levels of anti-toxin B IgG have been associated with milder disease. The development of high titers of anti-toxin A antibodies are associated with development of asymptomatic carriage, whereas low titers of anti-toxin A antibodies are associated with the subsequent development of CDI. Low levels of antibodies against toxin A have also been associated with more severe disease. Interestingly, up to 60% of healthy adults have detectable serum IgG and IgA against toxin A and toxin B, but the neutralizing effects of these antibodies wanes in the elderly. Thus, while there are clear correlations between protection against CDI and the presence of neutralizing antibodies, the reasons for loss of these responses with age remain unclear. This is due to the current lack of understanding of what constitutes a strong neutralizing response (i.e. which epitopes matter in a neutralizing response), which makes it difficult to study anti-toxin responses in humans.
Information on neutralizing antibody responses in humans could provide a powerful prognostic tool. It is unclear why some patients will develop severe infection when exposed to a pathogenic strain of C. difficile, while others only experience mild-diarrhea. It is very likely that neutralizing antibodies are a critical determinant in disease severity. By further analyzing the immune response present in patients with infection we hope to understand which antibodies are protective of severe or relapsing disease. Preliminary data shows that a majority of patients do indeed make antibodies against toxin B, but many of these “Ab-positive” patients do not produce neutralizing antibodies. Moreover, it has been shown that some patients make antibodies that neutralize toxin B from a hypervirulent strain of C. difficile but do not neutralize toxin B from a historical strain of C. difficile. In support of this observation, it has also found been found in animal models that antibodies do not cross neutralize the two forms of toxin B. The fact that toxin B antibodies from historical and hypervirulent strains do not cross-neutralize raises several concerns. First, vaccines developed towards the historical toxin may not protect against the hypervirulent toxin. Second, therapeutic monoclonal antibodies may not cross protect. Third, acquired immunity by infection with a historical strain or hypervirulent strain may not protect against infection with the reciprocal strain. For these reasons, there is a critical need to better understand what constitutes a neutralizing anti-toxin response in C. difficile infected patients, and to develop a suitable anti-toxin that may be used to protected against CDI and CDAD. Embodiments of the claimed invention set forth herein provides the specific regions of toxin B that are antigenic in humans and animal models and are responsible for neutralization of toxin B. Other embodiments of the invention provide platforms for the expression of large quantities of the toxin B peptide fragments that are used to raise antibodies against toxin B.